Ceratonia siliqua, known as the carob, is considered to be of high nutritional value and of great economic significance due to its unique composition. The beneficial effects of carob against cancer, metabolic syndrome, diabetes, diarrhea, hyperlipidemia and gastro esophageal reflux disease are only a few of its therapeutic actions. Metabolomics-based analysis provides an ultimate tool, for the deciphering of nutritional intervention derived metabolic alterations. In the present study, 16 male Wistar rats were treated with carob powder for a 15-day period. Fecal and urine samples were collected at 5 time points (0, 1, 5, 10 and 15 days). By the applied HILIC-MS/MS method, 63 and 67 hydrophilic metabolites were detected in the fecal and urine samples, respectively, including amino acids, organic acids, sugars, vitamins and other endogenous compounds. A clear group separation based on fecal metabolome was observed after 1 day and 15 days treatment, while only a mild differentiation at day 1 was observed based on urine metabolome. Twenty-one fecal metabolites were responsible for the separation including amino acids and their derivatives, vitamins and organic acids. However, only 7 metabolites were altered in rat urine samples. Metabolic alterations in fecal samples could be attributed to physiological and biochemical adaptations derived from the nutritional intervention. Fecal targeted metabolomics were proven to be suitable for uplifting and highlighting such alterations.